The lab has continued studies on ribonucleases H. These enzymes are important for studying retroviral infections such as HIV as well as participation in therapy by means of antisense degradation of messenger RNA. In addition, these proteins occupy a unique niche in biochemical studies because of their ability to distinguish between RNA-DNA and RNA-RNA duplexes and degrade the RNA moiety of RNA-DNA hybrids. The details of this recognition and discrimination will advance our understanding of mechanisms by which proteins interact with nucleic acids. We have shown that two types of RNases H found in bacteria also are present in eukaryotes. These are important findings in light of their potential involvement in antisense drug therapy and for assessment of inhibitors of HIV RNase H on cell functions resulting from inhibition of cellular RNases H. We have also defined a non-RNase H domain present on one class of eukaryotic RNases H that binds to duplex RNAs, possibly as a means of regulation of RNase H activity. A common motif of 40 amino acids is present in all eukaryotic enzymes of this class including yeasts and human cells. Interestingly, a protein involved in translation of the polycistronic mRNA of cauliflower mosaic virus has the same motif and when placed in the context of the yeast RNase H, functions as an RNA duplex binding site. This points to recognition of duplex RNAs by each of these proteins. This non-RNase H domain provides an opportunity to define the sites on the protein, in both domains, that bind to RNA-DNA hybrids and, thereby, provide significant insight into protein nucleic acid interactions. RNase H of this class purified to homogeneity has the undesirable property of binding to various surfaces and can easily be lost. However, when duplex RNAs are included, the protein binds to them and remains soluble. This property can be exploited to determine the minimum size of duplex RNA necessary for binding and offers the potential for obtaining co-crystals of protein and nucleic acid for determination of the structure by X-ray crystallography.